We have identified a novel form of Charcot-Marie-Tooth disease (CMT) - the first family with a recessive mutation (E210X) in the NEFL gene, which encodes neurofilament light (NFL). Children homozygous for this mutation develop a progressive, severe, sensory and motor neuropathy with slow conduction well into the traditional "demyelinating range". We found that the mutant protein did not form intermediate filaments in cultured cells. The density of myelinated axons was markedly reduced, and the remaining axons were small and lacked intermediate filaments in a sural nerve biopsy from one patient by electron microscopy (EM). These findings indicate that the E210X mutation is likely a null allele, so that homozygous null alleles of NEFL cause a severe progressive neuropathy in humans. The latter conclusion was completely unexpected as the Nefl-null mice and quiver quail (which harbor a nonsense mutation in Nefl) were investigated and found not to have a neuropathy, in spite of the finding that myelinated PNS axons were devoid of neurofilaments (NFs) and failed to enlarge normally during development. This discrepancy prompted us to re-examine some "old" (at least one- year-old) Nefl-null mice with EM, which showed degenerating myelinated axons not only in peripheral nerves, but also in optic nerve and spinal cord, suggesting that NFL is required for the maintenance of myelinated axons in both PNS and CNS. However, these mice had been maintained an inbred line, so the possibility that other genes contributed to these pathological findings could not be excluded. To definitely examine this hypothesis, we propose to back crossing the mice to their former background (C57/Bl6) and compare age- matched Nelf -/-, Nelf , and Nelf littermates by examining the L4 and L5 ventral roots, sciatic, femoral and optic nerves, and cervical and lumbar spinal cord by light microscopy, immunocytochemistry and EM at 3, 6, 12, 18 and 24 months of age, with the following Specific Aims: 1) Determine the timing and pattern of axonal degeneration in the PNS of Nefl-null mice. 2) Investigate the cellular basis of slowed conduction in the PNS of Nefl-null mice. 3) Determine the timing and pattern of axonal degeneration in the CNS of Nefl-null mice. These data will establish whether NFs are essential for the maintenance of myelinated axons in both PNS and CNS;such a role has not been demonstrated in prior studies. If evidence of axonal degeneration is found, this model can be used for further investigation to determine why neurofilaments are required for axonal integrity and to understand the mechanism of axonal degeneration in these patients and perhaps patients with other kinds of CMT. PUBLIC HEALTH RELEVANCE: The proposed study will establish whether NFs are essential for the maintenance of myelinated axons in both PNS and CNS;such a role has not been demonstrated in prior studies. If evidence of axonal degeneration is found, this model can be used for further investigation to determine why neurofilaments are required for axonal integrity and to understand the mechanism of axonal degeneration in these patients and perhaps patients with other kinds of CMT.